The transforming growth factor beta (TGF-.beta.) family has been implicated in a variety of physiological processes involving bone in vivo and in vitro. For example, in in vitro studies, TGF-.beta. stimulates proliferation and matrix synthesis of osteoblastic cells (Centrella, et al. (1987) J. Biol. Chem. 262:2869-2874) and has been reported to inhibit the formation and activity of osteoclastic cells (Chenu, et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:683-5687; Kiebzak et al. (1988) J. Bone Min. Res. 3:439-446). In vivo, subperiosteal injections of TGF-.beta. in the femur (Joyce, et al. (1990) J. Cell. Biol. 110:2195-2207) and calvarium (Noda and Camilliere (1989) Endocrinology 124:2991-2294) of rodents stimulate local bone formation. Furthermore, mice injected daily subcutaneously with TGF-.beta. showed an increase in the number of cuboidal osteoblasts along with increased deposition of bone matrix, while rats similarly treated showed an increase in osteoblasts, demonstrating the systemic in vivo activity of TGF-.beta. on bone (Matthews, et al. "Systemic delivery of TGF-.beta. produces a marked increase in osteoblasts, stimulates osteoid synthesis and increases bone formation in long bones and vertebrae in rats and mice," poster presented at the 1990 Meeting of the American Society of Bone and Mineral Research).
Activins are dimeric proteins structurally similar to inhibin, TGF-.beta.1, TGF-.beta.2, and other proteins that makeup a family of proteins structurally related to TGF-.beta.1. These proteins exhibit the chromatographic properties of TGF-.beta.s. In addition to having homology with respect to the amino acid sequences, activins exhibit conservation of cysteine positions characteristic of the TGF-.beta.s. Activins exhibit a molecular weight of 25 kD under nonreducing conditions by SDS-PAGE (and a molecular weight of 14 kD under reducing conditions). There are two known forms of the activin subunits, which have been termed .beta.A or .beta.B. Homodimeric forms .beta.AA and .beta.BB and a heterodimeric form .beta.AB have been described in the literature. Activin subunits have about a 30% homology to TGF-.beta.1 and TGF-.beta.2 chains in terms of their amino acid sequences. Inhibins are polypeptides which are also structurally related to activins. Inhibins are heterodimers of the activin .beta.A or .beta.B subunit and a separate .alpha. subunit. Inhibins exhibit activity essentially opposite to activin.
The activin .beta.A homodimer and .beta.AB heterodimer have been purified from porcine follicular fluid, and have been shown to stimulate the release of follicle stimulating hormone (FSH) from rat pituitary cells in vitro (W. Vale et al., Nature (1986) 321:776-79). Other reported activities include stimulation of oxytocin release from neurosecretory neurons (P. E. Sawchemko, et al., Nature (1988) 334:615-17; W. Vale et al., "Recent Progress in Hormone Research" (1988) 44:1-34); stimulation of insulin secretion from pancreatic islets (Y. Totsuka et al., Biochem. & Biophys. Res. Comm. (1988) 156:335-39); and stimulation of erythroid and multipotential progenitor cell colony formation in bone marrow culture (J. Yu et al., Nature (1987) 330:765-67; H. E. Broxmeyer et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85:9052-56). Activin .beta.A is apparently identical to erythroid differentiation factor (EDF) (M. Murata et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85:2434-38).
Despite the fact that activin is similar in amino acid sequence to TGF-.beta., activin does not compete with TGF-.beta. for binding to TGF-.beta. receptors types I, II, or III present on fibroblasts and epithelial cells. However, activin has been reported to compete against binding of TGF-.beta.1 to rat pituitary tumor cells (S. Cheifetz et al., J. Biol. Chem. (1988) 263:17225-28). TGF-.beta.1 and TGF-.beta.2 have been reported to induce formation of endochondral bone in vivo (M. E. Joyce et al., J. Cell Biol. (1990) 110:2195-2207, H. Bentz, et al. (1989) J. Biol. Chem., 264:20805-10).
A "bone morphogenetic protein" (BMP) was extracted from demineralized bone using urea or guanidine hydrochloride and reprecipitated according to the disclosures in U.S. Pat. Nos. 4,294,753 and 4,455,256 to Urist. Urist subsequently reported (Urist, M. R., Clin. Orthop. Rel. Res. (1982) 162:219) that ion exchange purification of this crude protein mixture yielded an activity which was unadsorbed to carboxymethyl cellulose resin (CMC) at pH 4.8. Urist's reports in Science (1983) 220:680-685 and Proc. Natl. Acad. Sci. U.S.A. (1984) 81:371-375 describe BMPs having molecular weights of 17,500 and 18,500 daltons. Urist's patent publication, EPA Publication No. 0212474, describes BMP fragments of 4,000 to 7,000 daltons obtained by limited proteolysis of BMP.
U.S. Pat. No. 4,608,199 describes a bone-derived protein of 30,000-32,000 daltons. The protein is described as being water soluble and having no affinity for concanavalin A.
WO 88/00205 reports four proteins, designated BMP-1, BMP-2 Class I ("BMP-2"), BMP-3, and BMP-2 Class II ("BMP-4"), that are alleged to have osteogenic activity.
J. M. Wozney, in Growth Factor Research, Vol. 1 (1989), pp. 267-280, describes three additional BMP proteins closely related to BMP-2, and which have been designated BMP-5, BMP-6 and BMP-7.
WO 89/09787 and 89/09788 describe a protein called "OP-1", now known to be BMP-7. The cloning of BMP-7 is described in E. Ozkaynak et al., EMBO Journal (1990) 9:2085-2093, and the purification of BMP-7 is described in T. K. Sampath et al., J. Biol. Chem. (1990) 265:13198-13205.
U.S. Pat. No. 4,434,094 to Seyedin and Thomas reported the partial purification of a bone generation-stimulating, bone-derived protein by extraction with chaotropic agents, fractionation on anion and cation exchange columns, and recovery of the activity from a fraction adsorbed to CMC at pH 4.8. This new protein fraction was termed "osteogenic factor" and was characterized as having a molecular weight below about 30,000 daltons.
Isgaard, et al. (Endocrinol. Metab. 13:E367-72,1986) reports the stimulation of bone growth by insulin-like growth factor (IGF).
Slovik et al. (J. Bone & Min. Res. 1:377-381, 1986) report the stimulation of bone growth by parathyroid hormone (PTH).
In vitro evidence suggests resorption of bone and formation of new bone appear to be coupled in some fashion. TGF-.beta., for example, is apparently involved in this process. Pfeilschifter and Mundy, Proc. Natl. Acad. Sci. U.S.A. (1987) 84:2024-2028), demonstrated that calvariae incubated with parathyroid hormone, 1,25-dihydroxyvitamin D.sub.3, and interleukin 1, all factors that stimulate bone resorption, showed an increase in endogenous TGF-.beta. activity in the culture medium, whereas incubation with calcitonin, which inhibits bone resorption, correlated with a decrease in endogenous TGF-.beta. activity.
The present invention offers in vivo combination therapy for stimulating new bone formation through the combined administration of an exogenous bone growth factor and an agent which inhibits bone resorption. These combinations provide more effective therapy for prevention of bone loss and replacement of bone than the components alone, as the combination appears to synergistically enhance the increase in bone mass and the rate of bone formation.